Acute Respiratory Distress Syndrome (ARDS) is a complication of serious infections and trauma and an important source of morbidity and mortality in Veterans and the general population. Despite improvements in supportive care, no pharmaceutical therapies have been developed and mortality remains over 40%. Although preclinical studies support pharmacologic inhibition of p38 mitogen-activated protein kinase (MAPK) to treat a range of inflammatory diseases, including ARDS, the currently available p38 inhibitors have had limited success in clinical trials due to toxicity and poor efficacy. Since these agents are catalytic site inhibitors, they block all p38 activity. Most of these inhibitors target multiple p38 isoforms, even though p38? is the only isoform required for inflammatory responses and other isoforms can be cytoprotective. Furthermore, since p38? itself also regulates anti-inflammatory and counterregulatory genes via Mitogen- and Stress-activated Protein Kinase (MSK)-1/2, we believe that targeting the catalytic site is an intrinsical- ly flawed strategy. We used computer-aided drug design (CADD) to identify novel small molecules that target the glutamate- aspartate (ED) substrate docking site, which is required for phosphorylation of MAPK-activated protein kinase-2 (MK2), a p38? substrate known to mediate inflammation, endothelial barrier function, and leukocyte mobility; whereas MSK1/2 binds to the c o m m o n d o c k i n g ( CD) site. Using this strategy, we identified p38?-binding compounds, including a lead compound, UM101, which selectively binds the CADD-targeted pocket in p38?, but not p38?. UM101 stabilizes human lung microvascular endothelial (HMVECL) barrier function, and inhibits LPS-induced proinflammatory gene expression in THP1 cells. UM101 is also well-tolerated and more potent than the prototypical p38 catalytic inhibitor, SB203580, in mitigating LPS-induced lung inflammation and permeability pulmonary edema in mice. RNASeq and Ingenuity Pathway Analysis showed that UM101 inhibited only some SB203580-inhibitable genes, but spared others including MSK1/2-dependent anti-inflammatory genes. Preliminary phosphopeptide analysis failed to identify off-target effects. Three additional compounds were identified from the CADD search, which share p38a-binding and biological activity with UM101. Our central hypothesis is that targeting small molecules to p38? substrate docking sites rather than the catalytic site will generate a spectrum of potential drugs with unique biological effects and reduced toxicity profiles, which will be more effective in treating inflammatory diseases. The objectives of this proposal are to: identify additional lead compounds with improved anti-inflammatory and endothelial barrier stabilizing activity, analyze their function and mechanism of action, and analyze toxicity and efficacy of at least three lead compounds as pre- and post-treatment in a mouse LPS-induced acute lung injury model. We will identify additional lead compounds by completing functional and mechanistic analyses of our 150 CADD- identified p38a-binding compounds. We expect to: (1) generate a series of chemically diverse, novel p38? modifiers that avoid the intrinsic flaw in currently available p38 catalytic inhibitors; (2) complete sufficient functional analysis to advance the lead compounds to chemical optimization to improve drug- like properties; and ( 3 ) c o m p le t e sufficient preclinical in vivo testing to support transition to clinical trials.